Primary and acquired resistance to the breast cancer drug trastuzumab (Herceptin) is a significant clinical problem. Here, we report enhanced activation of downstream signaling pathways emanating from the growth factor receptors erbB2, erbB3, and insulin-like growth factor-I receptor (IGF-IR) in trastuzumab-resistant breast cancer cells. Interactions between IGF-IR and erbB2 or erbB3 occur exclusively in trastuzumab-resistant cells, where enhanced erbB2-erbB3 interactions are also observed. Moreover, these three receptors form a heterotrimeric complex in resistant cells. erbB3 or IGF-IR knockdown by short hairpin RNA–mediated strategies upregulates p27^kip1, inactivates downstream receptor signaling, and resensitizes resistant cells to trastuzumab. Our findings reveal a heterotrimer complex with a key role in trastuzumab resistance. On the basis of our results, we propose that trastuzumab resistance in breast cancer might be overcome by therapeutic strategies that jointly target erbB3, erbB2, and IGF-IR. Cancer Res; 70(3); 1204–14